Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Distribution of paired immunoglobulin-like receptor B in the nervous system related to regeneration difficulties after unilateral lumbar spinal cord injury

Paired immunoglobulin-like receptor B（Pir B） is a functional receptor of myelin-associated inhibitors for axonal regeneration and synaptic plasticity in the central nervous system, and thus suppresses nerve regeneration. The regulatory effect of Pir B on injured nerves has received a lot of attention. To better understand nerve regeneration inability after spinal cord injury, this study aimed to investigate the distribution of Pir B（via immunofluorescence） in the central nervous system and peripheral nervous system 10 days after injury. Immunoreactivity for Pir B increased in the dorsal root ganglia, sciatic nerves, and spinal cord segments. In the dorsal root ganglia and sciatic nerves, Pir B was mainly distributed along neuronal and axonal membranes. Pir B was found to exhibit a diffuse, intricate distribution in the dorsal and ventral regions. Immunoreactivity for Pir B was enhanced in some cortical neurons located in the bilateral precentral gyri. Overall, the findings suggest a pattern of Pir B immunoreactivity in the nervous system after unilateral spinal transection injury, and also indicate that Pir B may suppress repair after injury.

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1（TRPV1） provides the sensation of pain（nociception）. However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517（300 mg/kg）, a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.

Effects of dexmedetomidine combined with ultrasound-guided nerve block on hemodynamics, immune function and cortisol levels in patients undergoing inguinal surgery

Objective:To investigate the effect of dexmedetomidine combined with ultrasound guided nerve block on the hemodynamics, immune function and cortisol level in the patients undergoing inguinal surgery.Methods:A total of 120 patients who underwent unilateral inguinal surgery in our hospital were randomly divided into control group and observation group, 60 cases in each group. The control group was induced by intravenous anesthesia with sufentanil. The observation group was given dexmedetomidine combined with ultrasound guided ilioinguinal/iliac hypogastric nerve block. Hemodynamics, immune function and cortisol levels were compared between the two groups before and after anesthesia induction. Results:After anesthesia, in surgical incision and recovery time, the levels of HR in the two groups were significantly lower than that before the anesthesia, and the levels of HR in the observation group were significantly lower than that in the control group. After anesthesia, in surgical incision and recovery time, the levels of CD3+, CD4+ and CD4+/CD8+ in the control group were significantly lower than that of the group before anesthesia. After anesthesia, the levels of CD3+, CD4+ and CD4+/CD8+ in the observation group were significantly lower than that of the group before anesthesia. In surgical incision and recovery time, there was no significant difference in the levels of CD3+, CD4+ and CD4+/CD8+ in the observation group before anesthesia. After anesthesia, in surgical incision and recovery time, the levels of CD3+, CD4+ and CD4+/CD8+ in the observation group were significantly higher than that of the control group. The levels of serum PI3K, ET -1, CRP and cortisol of the two groups were significantly higher than those before anesthesia, and the levels of PI3K, ET -1, CRP and cortisol in the serum of the patients in the observation group were significantly lower than those in the control group.Conclusion: The effect of dexmedetomidin combined with ultrasound guided nerve block anesthesia on the patients with inguinal surgery can effectively maintain the hemodynamic stability of the patients and have less influence on the immune function and the stress response. The security is higher.

Functional microglia derived from human pluripotent stem cells empower retinal organs

Microglia are known to play essential roles in the development,progression and treatment of diverse neurodegenerative diseases in the central nervous system,including the retina,brain and spinal cord.Recently,brain-induced microglia-like cells(iMGs)have been generated from human pluripotent stem cells(hPSCs);however,retinal microglia have yet to be developed in vitro.In this study,by mimicking in vivo microglial development,we established a simplified approach to differentiate hPSCs into high purity(>90%)iMGs.The iMGs express microglia-specific markers,release cytokines upon stimulation,and are capable of phagocytizing bacteria.When co-cultured with three-dimensional human retinal organoids(hROs),iMGs migrated into the hROs,tended to differentiate into resident retinal microglia,and simultaneously induced apoptosis in some neural cells.Notably,the resident i MGs in the hROs formed sparse web-like structures beneath the photoreceptor cell layer,resembling microglia's orientation in human retina.In conclusion,we developed a simplified and efficient method to generate microglia from human pluripotent stem cells,and we report the first derivation of retinaresident microglia in vitro,providing a new source of human retinal microglia for developmental and disease studies and regenerative therapeutics.